CN116478309A - Cynomorium songaricum polysaccharide, cynomorium songaricum polysaccharide compound, and preparation method and application thereof - Google Patents
Cynomorium songaricum polysaccharide, cynomorium songaricum polysaccharide compound, and preparation method and application thereof Download PDFInfo
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- CN116478309A CN116478309A CN202310574630.9A CN202310574630A CN116478309A CN 116478309 A CN116478309 A CN 116478309A CN 202310574630 A CN202310574630 A CN 202310574630A CN 116478309 A CN116478309 A CN 116478309A
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Classifications
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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Abstract
The invention provides cynomorium songaricum refined polysaccharide, a cynomorium songaricum polysaccharide compound, a preparation method and application thereof, and belongs to the technical field of medicines. The invention adopts ethanol water solution to extract cynomorium songaricum, and the obtained extract is concentrated to obtain ethanol extract; degreasing the alcohol extract by petroleum ether to obtain degreased residues; mixing the degreasing slag with water, standing, carrying out macroporous resin column chromatography separation on the obtained supernatant to finally obtain cynomorium songaricum crude polysaccharide, deproteinizing to obtain cynomorium songaricum refined polysaccharide, and further separating and purifying to obtain a cynomorium songaricum polysaccharide compound. The cynomorium songaricum refined polysaccharide and the cynomorium songaricum polysaccharide compound have high inhibition activity to type 5 phosphodiesterase, good selectivity, natural plant polysaccharide and low toxic and side effect and low price.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to cynomorium songaricum refined polysaccharide, a cynomorium songaricum polysaccharide compound, a preparation method and application.
Background
The active ingredients of the traditional Chinese medicine are always the main sources for developing new medicines, such as artemisinin, berberine, taxol and other medicines are discovered based on the traditional efficacy of the traditional Chinese medicine. The herba Cynomorii is dry fleshy stem of herba Cynomorii (Cynomorium songaricum rupr.) of Cynomoriaceae, has effects of invigorating kidney yang, replenishing essence and blood, loosening bowel to relieve constipation, and can be used for treating kidney yang deficiency, essence and blood deficiency, waist and knee flaccidity, sexual impotence, spermatorrhea and constipation due to intestinal dryness. Modern pharmacological activity researches show that cynomorium songaricum has various activities of resisting anoxia, resisting fatigue, inhibiting platelet aggregation, inhibiting glucocorticoid, regulating immunity, improving sexual function, improving intestinal function, promoting adrenal cortex secretion, resisting gastric ulcer, resisting transcription, resisting cancer and the like. However, the research on the action mechanism of cynomorium songaricum is relatively lacking, and the active ingredients for treating the diseases in cynomorium songaricum are not reported. The components of cynomorium songaricum mainly comprise organic acids, flavonoids, steroids, triterpenes, polysaccharides, amino acid compounds, mineral elements and the like, wherein the cynomorium songaricum polysaccharide is the most studied component at present, and the cynomorium songaricum polysaccharide mainly comprises the research on the aspects of treating diabetes, immunosuppression, antioxidation, aging resistance, improving gastrointestinal tract functions and the like. However, these studies lack reports on the "treating impotence and spermatorrhea" associated with the traditional efficacy of cynomorium songaricum. In the prior art, cynomorium songaricum polysaccharide is used for treating impotence after being combined with other various traditional Chinese medicines (such as Chinese patent with application number of CN 20181036830.5), but the invention is a traditional Chinese medicine composition, the mechanism of treating impotence is the result of the combined action of various components, and the invention does not clarify that the cynomorium songaricum polysaccharide is the main medicinal effect component in the traditional Chinese medicine composition. In addition, there is no report of treating impotence by monomeric polysaccharides in cynomorium songaricum polysaccharide.
In a human body, messenger molecule cyclic guanosine monophosphate (cGMP) promotes calcium ion outflow in smooth muscle cells to cause smooth muscle to loosen by controlling calcium ion channel Protein Kinase G (PKG), thereby causing blood inflow of pulmonary artery blood vessels, cavernous smooth muscle blood vessels, cerebral blood vessels and the like, and realizing physiological activities such as pulmonary artery pressure reduction, physiological erection, cerebral blood supply improvement and the like. Whereas cGMP acts as a substrate for phosphodiesterase type 5 and is readily degraded to Guanylate (GMP) and becomes inactive. Phosphodiesterase 5 is widely distributed in the lungs, corpora cavernosa smooth muscle of the human body, and tissues such as blood vessels, viscera, airway smooth muscle, skeletal muscle, platelets, etc. Thus, inhibitor molecules targeting phosphodiesterase type 5 have been used clinically in the treatment of pulmonary arterial hypertension and erectile dysfunction such as sildenafil, tadalafil, and the like. However, such synthetic chemicals often cause side effects in clinical treatment such as headache, facial flushing, dyspepsia, muscle pain, nasal obstruction, diarrhea, dizziness, rash, etc. In addition, patients who treat pulmonary hypertension need to take medicines for a long time, and the price of the medicines used clinically at present is generally high, so that the economic pressure of the patients is high. Except synthetic chemical medicines, the type 5 phosphodiesterase inhibitor of traditional Chinese medicine sources reported at present is only baohuoside compounds and analogues derived from epimedium, and is not widely applied clinically. It is therefore of great importance to find new phosphodiesterase type 5 inhibitors.
Disclosure of Invention
The invention aims to provide cynomorium songaricum polysaccharide, a cynomorium songaricum polysaccharide compound, a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of cynomorium songaricum refined polysaccharide, which comprises the following steps:
extracting cynomorium songaricum by adopting an ethanol water solution, and concentrating the obtained extracting solution to obtain an ethanol extract;
degreasing the alcohol extract by petroleum ether to obtain degreased residues;
mixing the degreasing slag with water, standing, separating the obtained supernatant by macroporous resin column chromatography, sequentially using 10% and 20-80% ethanol water solution as eluent, collecting eluent obtained when eluting the 20-80% ethanol water solution, sequentially concentrating and drying to obtain cynomorium songaricum crude polysaccharide;
deproteinizing the cynomorium songaricum crude polysaccharide to obtain cynomorium songaricum refined polysaccharide.
Preferably, the volume fraction of the ethanol aqueous solution used for extraction is 70-90%; the extraction is carried out under the condition of reflux, the times of the extraction are 2 to 5 times, the time of each extraction is 1.5 to 5 hours, and the feed liquid ratio of each extraction is 1g: 5-20 mL.
Preferably, the degreasing is carried out under boiling conditions, the times of degreasing are 3-8 times, the time of each degreasing is 10-60 min, and the ratio of the degreasing liquid to the degreasing liquid is 1g: 5-10 mL.
Preferably, macroporous adsorption resin adopted by macroporous resin column chromatographic separation is nonpolar macroporous polystyrene adsorption resin.
The invention provides cynomorium songaricum refined polysaccharide prepared by the preparation method.
The invention provides a cynomorium songaricum polysaccharide compound, which has a linkage mode shown in a formula V:
the invention provides a preparation method of the cynomorium songaricum polysaccharide compound, which comprises the following steps:
taking water as an eluent, adopting a cellulose column to carry out polarity separation on cynomorium songaricum refined polysaccharide in the technical scheme, and sequentially concentrating, dialyzing and drying the obtained eluent to obtain a water eluting component;
and (3) purifying the water elution component by using a sephadex column with a sodium chloride aqueous solution with the concentration of 0.1-0.3 mol/L as an eluent to obtain the cynomorium songaricum polysaccharide compound with the linkage mode shown in the formula V.
The invention provides a cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or application of the cynomorium songaricum polysaccharide in preparation of a type 5 phosphodiesterase inhibitor, wherein the cynomorium songaricum polysaccharide compound has a linkage mode shown in a formula I, a formula II, a formula III, a formula IV or a formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
the invention provides a cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or application of the cynomorium songaricum polysaccharide in preparation of medicaments for preventing and treating diseases related to phosphodiesterase type 5 of human or animal, wherein the cynomorium songaricum polysaccharide compound has a linking mode shown in formula I, formula II, formula III, formula IV or formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
preferably, the disease associated with phosphodiesterase type 5 comprises pulmonary hypertension, male erectile dysfunction, male testicular ischemia, female sexual dysfunction, dysmenorrhea, benign prostatic hyperplasia, ischemic angina, hypertension, alzheimer's disease, arteriosclerosis, stroke, arteriosclerotic occlusion, arterial embolism, chronic asthma, bronchitis, allergic asthma, allergic rhinitis, glaucoma or a disease characterized by intestinal motility disorder.
The invention provides a preparation method of cynomorium songaricum refined polysaccharide, which comprises the following steps: extracting cynomorium songaricum by adopting an ethanol water solution, and concentrating the obtained extracting solution to obtain an ethanol extract; degreasing the alcohol extract by petroleum ether to obtain degreased residues; mixing the degreasing slag with water, standing, separating the obtained supernatant by macroporous resin column chromatography, sequentially using 10% and 20-80% ethanol water solution as eluent, collecting eluent obtained when eluting the 20-80% ethanol water solution, sequentially concentrating and drying to obtain cynomorium songaricum crude polysaccharide; deproteinizing the cynomorium songaricum crude polysaccharide to obtain cynomorium songaricum refined polysaccharide; the cynomorium songaricum polysaccharide compound can be further separated and purified on the basis of the method. The cynomorium songaricum refined polysaccharide and the cynomorium songaricum polysaccharide compound have high inhibition activity to type 5 phosphodiesterase, good selectivity and small toxic and side effects, and are natural plant polysaccharide, wherein the content of cynomorium songaricum crude polysaccharide in cynomorium songaricum is up to 10%, so that resources are guaranteed for future clinical application, the price of type 5 phosphodiesterase inhibitor can be reduced, and the problem of high price of the current medicament is solved.
Detailed Description
The invention provides a preparation method of cynomorium songaricum refined polysaccharide, which comprises the following steps:
extracting cynomorium songaricum by adopting an ethanol water solution, and concentrating the obtained extracting solution to obtain an ethanol extract;
degreasing the alcohol extract by petroleum ether to obtain degreased residues;
mixing the degreasing slag with water, standing, separating the obtained supernatant by macroporous resin column chromatography, sequentially using 10% and 20-80% ethanol water solution as eluent, collecting eluent obtained when eluting the 20-80% ethanol water solution, sequentially concentrating and drying to obtain cynomorium songaricum crude polysaccharide;
deproteinizing the cynomorium songaricum crude polysaccharide to obtain cynomorium songaricum refined polysaccharide.
In the present invention, the raw materials used are commercial products well known to those skilled in the art unless specified otherwise; the water used is distilled water.
The invention adopts ethanol aqueous solution to extract cynomorium songaricum, and the obtained extract is concentrated to obtain an ethanol extract. In the invention, the cynomorium songaricum is preferably dried and crushed sequentially before being extracted; the drying is preferably air drying; the comminution is preferably based on obtaining cynomorium songaricum particles having a particle size of 10 mesh. In the present invention, the volume fraction of the aqueous ethanol solution used for the extraction is preferably 70 to 90%, more preferably 80 to 90%; the extraction is preferably carried out under reflux conditions, the specific temperature being preferably 75 ℃; the number of the extractions is preferably 2 to 5, more preferably 3; the time of each extraction is preferably 1.5 to 5 hours, more preferably 2 to 3 hours; the ratio of feed liquid extracted each time is preferably 1g:5 to 20mL, more preferably 1g: 7-10 mL. The invention preferably combines the extracting solutions obtained by each extraction and then concentrates the extracting solutions; the concentration is preferably reduced pressure concentration; the concentration is preferably such that the extract is concentrated to no fluidity of the sample. The invention preferably extracts under the conditions, and can extract most polysaccharides with different water solubility compared with the traditional water extraction method, thereby being beneficial to more completely extracting cynomorium songaricum polysaccharide components.
After the alcohol extract is obtained, petroleum ether is adopted to degrease the alcohol extract, so that degreased residues are obtained. In the present invention, the degreasing is preferably performed under boiling conditions; the number of times of degreasing is preferably 3 to 8 times, more preferably 5 times; the time for each degreasing is preferably 10 to 60 minutes, more preferably 20 to 30 minutes; the ratio of the degreasing liquid to the degreasing liquid is preferably 1g:5 to 10mL, more preferably 1g: 5-7 mL. After degreasing, the obtained material is preferably subjected to solid-liquid separation, the obtained liquid material contains fat compounds, and the obtained solid material is the degreasing slag. The degreasing is preferably carried out under the conditions, so that lipid components can be effectively removed, and the subsequent separation and purification of cynomorium songaricum polysaccharide components are facilitated.
After obtaining the degreasing slag, the invention mixes the degreasing slag with water and stands, the obtained supernatant is subjected to macroporous resin column chromatography separation, the eluent is ethanol water solution with the volume fraction of 10 percent and 20-80 percent in sequence, the eluent obtained when the ethanol water solution with the volume fraction of 20-80 percent is collected is eluted, and the concentration and the drying are carried out in sequence, so that the cynomorium songaricum crude polysaccharide is obtained. In the invention, the dosage ratio of the degreasing slag to the water is preferably 1g:5 to 20mL, more preferably 1g:10mL. In the present invention, the temperature of the rest is preferably 10 to 30 ℃, more preferably 20 ℃; the time is preferably 1 to 5 hours, more preferably 2 hours. The invention mixes the degreasing slag with water for standing to remove non-saccharide small polar components mixed in the extraction process.
After the standing, the invention takes supernatant liquid to carry out macroporous resin column chromatographic separation. In the invention, the macroporous resin column is preferably balanced by water before use; the specific manner of balancing is not particularly limited, and may be any manner known to those skilled in the art. In the invention, the macroporous adsorption resin adopted by macroporous resin column chromatographic separation is preferably nonpolar macroporous polystyrene adsorption resin, more preferably macroporous adsorption resin with the model of Amberlite XAD1600N, XAD N or XAD1180N, and further preferably macroporous adsorption resin with the model of Amberlite XAD 1600N. The invention preferably adds the supernatant into a macroporous resin column which is balanced by water in advance, adopts an ethanol water solution with the volume fraction of 10 percent to carry out first elution, then adopts an ethanol water solution with the volume fraction of 20-80 percent to carry out second elution, collects eluent obtained during the second elution, and sequentially carries out concentration and drying to obtain cynomorium songaricum crude polysaccharide. In the present invention, the volume fraction of the aqueous ethanol solution used for the second elution is preferably 50 to 75%, more preferably 65 to 70%. In the present invention, the drying is preferably freeze-drying. The macroporous resin column chromatography separation is preferably carried out under the conditions, so that the main polysaccharide component can be rapidly separated from the supernatant, and compared with the traditional water boiling method, the method has the advantages of high speed and partial free protein removal, and is beneficial to industrial production. After the second elution, the invention preferably adopts an ethanol water solution with the volume fraction of 95 percent to continue elution so as to fully remove impurities in the macroporous resin column and facilitate the next use.
After the cynomorium songaricum crude polysaccharide is obtained, deproteinizing the cynomorium songaricum crude polysaccharide to obtain cynomorium songaricum refined polysaccharide. In the invention, the deproteinization method is preferably a Sevage method; the Sevage method is not particularly limited, and may be any method known to those skilled in the art.
The invention provides cynomorium songaricum refined polysaccharide prepared by the preparation method. The cynomorium songaricum refined polysaccharide provided by the invention has high inhibition activity to type 5 phosphodiesterase, good selectivity, and is natural plant polysaccharide, and has the characteristics of small toxic and side effects and low price.
The invention provides a cynomorium songaricum polysaccharide compound, which has a linkage mode shown in a formula V:
in the invention, a cynomorium songaricum polysaccharide compound with a linkage mode shown in a formula V is denoted as CS-P5, wherein the CS-P5 is a polysaccharide formed by repeatedly connecting basic units consisting of 26 alpha-D-glucose (alpha-D-Glc), 8 beta-D-mannose (beta-D-Man), 11 beta-D-galactose (beta-D-Gal), 1 alpha-L-rhamnose (alpha-L-Rha) and 5 alpha-L-arabinose (alpha-L-Araf), and contains 3 branched chains. In the present invention, the CS-P5 has a weight average molecular weight of 9609.
The invention provides a preparation method of the cynomorium songaricum polysaccharide compound, which comprises the following steps:
taking water as an eluent, adopting a cellulose column to carry out polarity separation on cynomorium songaricum refined polysaccharide in the technical scheme, and sequentially concentrating, dialyzing and drying the obtained eluent to obtain a water eluting component;
and (3) purifying the water eluted component by using a sephadex column with a sodium chloride aqueous solution with the concentration of 0.1-0.3 mol/L as an eluent to obtain the cynomorium songaricum polysaccharide compound (CS-P5) with the linkage mode shown in the formula V.
According to the invention, water is used as an eluent, a cellulose column is used for carrying out polarity separation on cynomorium songaricum refined polysaccharide in the technical scheme, and the obtained eluent is sequentially concentrated, dialyzed and dried to obtain a water eluting component. In the present invention, the type of the filler in the cellulose column is preferably DEAE-52. In the present invention, the cellulose column is preferably soaked with hydrochloric acid before use to remove impurities in the packing, then eluted to neutrality with water, and equilibrated with water. In the present invention, the hydrochloric acid concentration used for the soaking is preferably 0.1 to 1mol/L, more preferably 0.5mol/L; the soaking time is preferably 0.5-10 h, more preferably 1h; in the process of eluting to neutrality, the volume of water used is preferably 4-10 times of the volume of the column, more preferably 5 times of the volume of the column; the flow rate of water used during the equilibration is preferably 3 to 20mL/min, more preferably 5mL/min, and the equilibration time is preferably 1 to 5 hours, more preferably 2 hours.
The invention preferably mixes the cynomorium songaricum refined polysaccharide with water, vortex after heating, then centrifugate, take supernatant fluid to load on the cellulose column which is well balanced by water, then use water as eluent to carry out elution. In the present invention, the heating temperature is preferably 30 to 50 ℃, more preferably 40 ℃; the rotating speed of the vortex is preferably 500-1500 rpm, more preferably 1000rpm, and the time is preferably 2-20 min, more preferably 5-10 min; the rotational speed of the centrifugation is preferably 10000 to 15000rpm, more preferably 12000rpm, and the time is preferably 5 to 20min, more preferably 5 to 10min. In the present invention, the effect of the heating is to increase the solubility of the poorly soluble polysaccharide; the invention preferably adopts the mode to obtain the supernatant and adopts the cellulose column to carry out polarity separation, so that the polysaccharide with different polarities can be separated, and the separation of the polysaccharide with different structure types is facilitated. In the present invention, the flow rate of water at the time of elution is preferably 5 to 50mL/min, more preferably 15mL/min; the amount of water to be used is preferably 1 to 10 times the column volume, more preferably 3 times the column volume. The invention preferably adopts a phenol sulfuric acid method for tracking detection, and collects eluent corresponding to symmetrical peaks; specifically, the eluent obtained during water elution is collected and sequentially concentrated, dialyzed and dried to obtain a water elution component (CS-P-W component). The concentration is not particularly limited, and may be performed by any concentration means known to those skilled in the art. In the present invention, the dialysis bag used for the dialysis preferably has a molecular weight cut-off of 3500Da, and the dialysis is preferably performed in pure water. In the present invention, the drying is preferably freeze-drying.
After the water elution component is obtained, the invention uses sodium chloride aqueous solution with the concentration of 0.1-0.3 mol/L as an eluent, and adopts a sephadex column to purify the water elution component to obtain CS-P5. In the present invention, the type of the packing in the sephadex column preferably includes Sepharose CL-6B, sepharose CL-4B or Sepharose CL-2B. In the present invention, the concentration of the aqueous sodium chloride solution used for the purification is 0.1 to 0.3mol/L, preferably 0.2mol/L. The invention preferably combines with an differential detector to perform online detection and collection, collects purified liquid corresponding to a symmetrical peak, and sequentially performs concentration and drying to obtain CS-P5; in an embodiment of the present invention, the apparatus for concentration is preferably a rotary evaporator; the drying is preferably freeze-drying. In the process of purifying the water elution component, another cynomorium songaricum polysaccharide compound is also obtained and is marked as CS-P1, and the linking mode of the CS-P1 is specifically shown as a formula I:
3) -alpha-D-Araf- (1- > 3) -alpha-D-Glcp- (1- > 4) alpha-D-GalpA 6Me- (1- > formula I.
In the present invention, CS-P1 is a polysaccharide in which three kinds of monosaccharides, namely, alpha-D-arabinose (alpha-D-Araf), alpha-D-glucose (alpha-D-Glc) and alpha-D-6' methyl galactose (alpha-D-GalA 6 Me), are repeatedly linked. In the present invention, the CS-P1 has a weight average molecular weight of 1394500.
In the invention, water is taken as an eluent, after the cynomorium songaricum polysaccharide in the technical scheme is subjected to polarity separation by adopting a cellulose column, aqueous NaCl solution with concentration of 0.1-0.3 mol/L (preferably 0.2 mol/L), 0.4-0.6 mol/L (preferably 0.5 mol/L) and 0.8-1.2 mol/L (preferably 1.0 mol/L) is preferably used as the eluent for continuous elution, and eluent obtained when the eluent with each concentration is collected is subjected to concentration, dialysis and drying in sequence, so as to obtain the eluted components of the aqueous NaCl solution; and then obtaining other kinds of cynomorium songaricum polysaccharide compounds by referring to the purification method. Specifically, in the embodiment of the invention, the elution components of the NaCl aqueous solution corresponding to the obtained eluent when the NaCl aqueous solution with the concentration of 0.2mol/L, 0.5mol/L and 1.0mol/L is used as the eluent are sequentially marked as a CS-P-0.2M component, a CS-P-0.5M component and a CS-P-1.0M component; then, the CS-P-0.2M component is subjected to the method of sephadex purification to obtain another cynomorium songaricum polysaccharide compound which is marked as CS-P2; the CS-P-0.5M component is subjected to the method of sephadex purification to obtain another cynomorium songaricum polysaccharide compound which is marked as CS-P3; the CS-P-1.0M fraction was subjected to the above-mentioned method of Sephadex purification to obtain another cynomorium songaricum polysaccharide compound, designated CS-P4.
In the invention, the linking mode of the CS-P2 is specifically shown as a formula II:
in the present invention, the CS-P2 is a polysaccharide formed by repeatedly linking basic units consisting of 5 alpha-D-glucose (alpha-D-Glc), 1 beta-D-glucose (beta-D-Glc), 1 alpha-D-mannose (alpha-D-Man) and 1 beta-D-galactose (beta-D-Gal). In the present invention, the CS-P2 has a weight average molecular weight of 482500.
In the invention, the linking mode of CS-P3 is specifically shown in the formula III:
α -Glcp- (1→6) - α -Glcp- (1→3) - α -Glcp- (1→6) - α -Glcp- (1→formula III.
In the present invention, CS-P3 is a polysaccharide in which the basic units consisting of 5 α -D-glucose (α -D-Glc) are repeatedly linked. In the present invention, the CS-P3 has a weight average molecular weight of 715000.
In the invention, the linking mode of the CS-P4 is specifically shown as a formula IV:
in the invention, the CS-P4 is a polysaccharide formed by repeatedly connecting basic units consisting of 6 alpha-D-arabinose (alpha-D-Araf), 2 beta-D-galactose (beta-D-Gal), 2 alpha-D-glucose (alpha-D-Glc) and 2 alpha-D-rhamnose (alpha-D-Rha), and contains 2 branched chains. In the present invention, the CS-P4 has a weight average molecular weight of 1470000.
The invention provides a cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or application of the cynomorium songaricum polysaccharide in preparation of a type 5 phosphodiesterase inhibitor, wherein the cynomorium songaricum polysaccharide compound has a linkage mode shown in a formula I, a formula II, a formula III, a formula IV or a formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
the cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or cynomorium songaricum polysaccharide in the technical scheme can inhibit type 5 phosphodiesterase and can be used for preparing type 5 phosphodiesterase inhibitors.
The invention provides a cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or application of the cynomorium songaricum polysaccharide in preparation of medicaments for preventing and treating diseases related to phosphodiesterase type 5 of human or animal, wherein the cynomorium songaricum polysaccharide compound has a linking mode shown in formula I, formula II, formula III, formula IV or formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
according to the invention, the cynomorium songaricum polysaccharide compound, pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, enantiomers or the cynomorium songaricum polysaccharide can inhibit type 5 phosphodiesterase, so that diseases or disease states related to type 5 phosphodiesterase can be eliminated, improved or relieved, and the diseases related to type 5 phosphodiesterase can be prevented and treated.
In the present invention, the pharmaceutically acceptable salt of the cynomorium songaricum polysaccharide compound includes a salt of the cynomorium songaricum polysaccharide compound with an inorganic base, preferably including sodium hydroxide or potassium hydroxide.
In the invention, the medicament comprises a pharmaceutically acceptable carrier, and the content of the pharmaceutically acceptable carrier in the medicament is preferably 1-98 wt%, more preferably 10-80 wt%. In the present invention, the pharmaceutically acceptable carrier preferably includes, but is not limited to: ion exchangers (e.g. Na may be + 、Ca 2+ 、K + 、Mg 2+ 、Fe 3+ The metal salt or metal base may be a sulfonic acid group (-SO) contained 3 H) Or carboxylic group (-COOH) acid group, aluminum oxide, aluminum stearate, lecithin, serum protein (such as human serum protein), buffer substance (such as phosphate buffer salt or HEPE)S buffer, etc.), glycerin, sorbitol, partial glyceride mixtures of saturated vegetable fatty acids, water, electrolytes (such as sulfuric acid or sodium chloride), protamine, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, starch, polyacrylate, beeswax and lanolin.
In the present invention, the drug may be administered in unit dosage form, and the dosage form of the drug is not particularly limited, and may be any dosage form known to those skilled in the art. The mode of administration of the drug is not particularly limited in the present invention, and may be any mode known to those skilled in the art depending on the dosage form of the drug.
In the present invention, the diseases associated with phosphodiesterase type 5 preferably include pulmonary hypertension, male erectile dysfunction, male testicular ischemia, female sexual dysfunction, dysmenorrhea, benign prostatic hyperplasia, ischemic angina, hypertension, alzheimer's disease, arteriosclerosis, stroke, arteriosclerotic occlusion, arterial embolism, chronic asthma, bronchitis, allergic asthma, allergic rhinitis, glaucoma or diseases characterized by intestinal motility disorder, more preferably pulmonary arterial hypertension or male erectile dysfunction. The invention can improve the local or whole body vascular state, especially the local or whole body arterial blood flow, through the corresponding administration mode, thus can be used for preventing and treating the diseases.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of cynomorium songaricum refined polysaccharide
The cynomorium songaricum medicinal material is dried and crushed to obtain cynomorium songaricum particles with the granularity of 10 meshes, ethanol water solution with the volume fraction of 90% is used for extracting for 3 times under the condition of reflux (75 ℃), the extracting time is 2 hours each time, and the ratio of feed liquid extracted each time is 1:10 (g/mL), combining the 3 times of extracting solutions, and concentrating under reduced pressure until the sample is free of fluidity to obtain an alcohol extract;
degreasing the alcohol extract with petroleum ether for 5 times under boiling conditions, wherein the degreasing time is 30min each time, and the degreasing feed liquid ratio is 1 each time: 5 (g/mL), then filtering, wherein the filtrate contains fatty compounds, and the filter residue and distilled water are mixed according to the weight ratio of 1g: mixing 10mL to obtain suspension, standing at 20deg.C for 2 hr, adding the supernatant into macroporous resin column (Amberlite XAD 1600N) with balanced distilled water, sequentially eluting with 10% ethanol water solution, 70% ethanol water solution and 95% ethanol water solution, collecting eluate obtained when 70% ethanol water solution is eluted, concentrating and lyophilizing to obtain herba Cynomorii crude polysaccharide;
deproteinizing the cynomorium songaricum crude polysaccharide by adopting a Sevage method to obtain cynomorium songaricum refined polysaccharide.
Example 2 isolation and characterization of CS-P1 to CS-P5
Performing polarity separation on cynomorium songaricum refined polysaccharide prepared in the embodiment 1 through a cellulose column (DEAE-52), wherein before the cellulose column is used, the DEAE-52 packing is soaked in 0.5M hydrochloric acid for 1h to remove impurities in the DEAE-52 packing, then distilled water with the volume of 5 times of column volume is eluted to be neutral under the condition that the flow rate is 15mL/min, and then the flow rate of distilled water is adjusted to be 5mL/min to be balanced for 2h; mixing cynomorium songaricum polysaccharide with distilled water, heating to 40 ℃, swirling for 5min at 1000rpm, centrifuging for 10min at 12000rpm, sampling supernatant, adjusting the flow rate of eluent to 15mL/min, eluting with distilled water, 0.2M NaCl water solution, 0.5M NaCl water solution and 1.0M NaCl water solution in sequence, wherein the dosage of each eluent is 3 times of column volume, carrying out tracking detection by using a phenol sulfuric acid method, collecting eluent corresponding to symmetrical peaks, dialyzing in pure water by using 3500Da dialysis bags after concentration, and obtaining components of CS-P-0.2M component, CS-P-0.5M component and CS-P-1.0M component (eluent obtained when eluting with distilled water, 0.2M NaCl water solution, 0.5M NaCl water solution and 1.0M NaCl water solution respectively);
purifying the CS-P-W component, the CS-P-0.2M component, the CS-P-0.5M component and the CS-P-1.0M component respectively by a dextran gel column (SUGAR-BRT-103), wherein the eluent is 0.2M NaCl aqueous solution, collecting the purified solution corresponding to a symmetrical peak by combining with an error detector for online detection, concentrating by a rotary evaporator, and freeze-drying to obtain compounds which are named as CS-P1, CS-P2, CS-P3, CS-P4 and CS-P5, wherein the CS-P-W component is purified to obtain CS-P1 and CS-P5, the CS-P-0.2M component is purified to obtain CS-P2, the CS-P-0.5M component is purified to obtain CS-P3, and the CS-P-1.0M component is purified to obtain CS-P4; the weight average molecular weights of CS-P1, CS-P2, CS-P3, CS-P4 and CS-P5 are respectively: 1394500, 482500, 715000, 1470000, 9609.
The assignment data of the carbon spectrum and the hydrogen spectrum of CS-P1, CS-P2, CS-P3, CS-P4 and CS-P5 are shown in tables 1 to 5.
TABLE 1 assignment of carbon and Hydrogen Spectrometry data for the Main fragment in CS-P1
TABLE 2 assignment of carbon and Hydrogen Spectrometry data for the Main fragment in CS-P2
TABLE 3 assignment of carbon and Hydrogen Spectrometry data for the Main fragment in CS-P3
TABLE 4 assignment of carbon and Hydrogen Spectrometry data for the Main fragment in CS-P4
TABLE 5 carbon and Hydrogen Spectrum data belonging to the main fragment of CS-P5 table->
EXAMPLE 3 biological Activity assay
1. In vitro inhibitory Activity against phosphodiesterase 5
In this example, the in vitro inhibitory activity of cynomorium songaricum polysaccharide and 5 polysaccharide compounds (compounds CS-P1-CS-P5) on phosphodiesterase type 5 (PDE 5) and other members of the phosphodiesterase family was measured by reference to literature method (A label-free LC/MS-based enzymatic activity assay forthe detection ofPDE5 Ainshibitors, front chem.2023, 13; 11:1097027), and the specific results are shown in Table 6, with the data in Table 6 being the average of at least 3 experiments. From table 6, cynomorium songaricum polysaccharide and 5 polysaccharide compounds can inhibit phosphodiesterase type 5, but have no inhibitory activity on other phosphodiesterase subtypes, indicating that cynomorium songaricum polysaccharide and 5 polysaccharide compounds have selective inhibitory activity on phosphodiesterase type 5.
TABLE 6 Cynomorium songaricum polysaccharide and in vitro inhibitory Activity of 5 polysaccharide Compounds against phosphodiesterase 5 data
2. Detection of cGMP levels in serum
42 SD rats were equally divided into 1 control group (n=6, physiological saline administration) and 6 administration groups (n=6), and each administration group was administered with cynomorium songaricum polysaccharide at a dose of 100mg/kg and 5 polysaccharide compounds (compounds CS-P1 to CS-P5) by intragastric administration. 1mL of orbital blood collection is placed in a sterilized centrifuge tube, and after standing for 1h, a serum sample is obtained by centrifugation, and then the content of cGMP is measured by using a kit.
Table 7 shows the change data of cGMP content in blood after taking cynomorium songaricum polysaccharide and 5 polysaccharide compounds, and the results show that the content of cGMP in serum of the rat can be remarkably improved after the cynomorium songaricum polysaccharide and 5 polysaccharide compounds are taken relative to a control group, so that metabolism of cGMP can be inhibited in the rat body by the cynomorium songaricum polysaccharide and 5 polysaccharide compounds, and the effect of PDE5 inhibitor is consistent.
TABLE 7 cGMP content in blood after administration of Cynomorium songaricum polysaccharide and 5 polysaccharide compounds to rats
3. Therapeutic effects on pulmonary arterial hypertension
The treatment activity of cynomorium songaricum polysaccharide and 5 polysaccharide compounds (compounds CS-P1 to CS-P5) is tested by establishing a hypoxia pulmonary artery high pressure model through a hypoxia induction method. The grouping method comprises the following steps: the average of 48 male SD rats was divided into 8 groups, specifically a normoxic control group (n=6), a model group (n=6), a model+cynomorium songaricum polysaccharide group (cynomorium songaricum polysaccharide and 5 polysaccharide compounds, n=6, at a dose of 100 mg/kg). The model building method comprises the following steps: SD rats were placed in a hypoxic chamber and fed under a pressure of 47.3kPa at a relative oxygen concentration of 10% for 2 weeks. Pulmonary artery pressure measurement method: the rats of each group are fixed on an operating table in a lying position after being anesthetized by intraperitoneal injection of pentobarbital sodium, the chest is opened after vital signs are stabilized, the lungs and the hearts are fully exposed, a hard polyethylene catheter filled with heparinized normal saline is rapidly inserted into the right ventricle, and the other end of the catheter is connected with a PowerLab data acquisition and analysis system to monitor pressure change.
Table 8 shows the pressure change data of rats after taking cynomorium songaricum polysaccharide and 5 polysaccharide compounds, and the results show that the pulmonary artery pressure of the rats is obviously increased after hypoxia induction, which indicates successful modeling, and the pulmonary artery pressure is obviously reduced after treatment by cynomorium songaricum polysaccharide and 5 polysaccharide compounds, which indicates that the cynomorium songaricum polysaccharide and 5 polysaccharide compounds have the activity of obviously reducing the pulmonary artery pressure.
Table 8 pressure data after rats dosed with cynomorium songaricum polysaccharide and 5 polysaccharide compounds
4. Activity for improving sexual function
The experiment mainly observes the change of the sexual behavior index of male rats after the male rats are given cynomorium songaricum polysaccharide and 5 polysaccharide compounds (compounds CS-P1-CS-P5), and mainly comprises the capture times (the times of capturing female rats from female rats after the female rats are put into a cage) and the climbing times (the times of climbing behaviors of the male rats after the female rats are put into the cage), and the specific experimental design is as follows: the 42 male SD rats are equally divided into 7 groups, specifically 1 control group (n=6, the same volume of physiological saline is added) and 6 administration groups (n=6, the dose is 100 mg/kg), wherein the administration groups are respectively prepared into aqueous solutions of cynomorium songaricum polysaccharide and 5 polysaccharide compounds and are administrated by one-time gastric lavage according to the administration amount of 10 mL/kg. And (3) 10min before the behavior observation experiment, firstly putting a male experiment rat into a behavior observation box for adaptation, opening a camera after the adaptation is finished, and then putting a female rat with estrus into the camera for shooting, wherein the time for behavior shooting is 1h.
Table 9 shows that the capturing times and the climbing times of the rats after taking the cynomorium songaricum polysaccharide and the 5 polysaccharide compounds are remarkably improved compared with the control group after taking the cynomorium songaricum polysaccharide and the 5 polysaccharide compounds, and the results show that the cynomorium songaricum polysaccharide and the 5 polysaccharide compounds have an improvement effect on sexual function.
TABLE 9 number of captures and climbs after rat took cynomorium songaricum polysaccharide and 5 polysaccharide compounds
| Group of | Number of captures | Number of climbing steps |
| Refined polysaccharide | 9.33±2.71 | 7.20±1.50 |
| CS-P1 | 12.04±1.85 | 9.14±1.45 |
| CS-P2 | 14.50±1.61 | 10.45±1.02 |
| CS-P3 | 15.52±2.70 | 12.82±0.98 |
| CS-P4 | 13.60±2.95 | 13.40±1.40 |
| CS-P5 | 16.84±2.26 | 13.23±1.30 |
| Control group | 7.89±1.59 | 5.05±0.80 |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A preparation method of cynomorium songaricum refined polysaccharide comprises the following steps:
extracting cynomorium songaricum by adopting an ethanol water solution, and concentrating the obtained extracting solution to obtain an ethanol extract;
degreasing the alcohol extract by petroleum ether to obtain degreased residues;
mixing the degreasing slag with water, standing, separating the obtained supernatant by macroporous resin column chromatography, sequentially using 10% and 20-80% ethanol water solution as eluent, collecting eluent obtained when eluting the 20-80% ethanol water solution, sequentially concentrating and drying to obtain cynomorium songaricum crude polysaccharide;
deproteinizing the cynomorium songaricum crude polysaccharide to obtain cynomorium songaricum refined polysaccharide.
2. The method according to claim 1, wherein the volume fraction of the aqueous ethanol solution used for the extraction is 70 to 90%; the extraction is carried out under the condition of reflux, the times of the extraction are 2 to 5 times, the time of each extraction is 1.5 to 5 hours, and the feed liquid ratio of each extraction is 1g: 5-20 mL.
3. The preparation method according to claim 1, wherein the degreasing is performed under boiling conditions, the number of times of degreasing is 3-8, the time of each degreasing is 10-60 min, and the ratio of the liquid to the solid to the liquid is 1g: 5-10 mL.
4. The method according to claim 1, wherein the macroporous resin used for the macroporous resin column chromatography separation is a nonpolar macroporous polystyrene adsorption resin.
5. The cynomorium songaricum polysaccharide prepared by the preparation method of any one of claims 1-4.
6. A cynomorium songaricum polysaccharide compound having a linkage pattern represented by formula V:
7. the method for preparing a cynomorium songaricum polysaccharide compound according to claim 6, comprising the following steps:
performing polarity separation on cynomorium songaricum refined polysaccharide according to claim 5 by using a cellulose column by using water as an eluent, and sequentially concentrating, dialyzing and drying the obtained eluent to obtain a water eluting component;
and (3) purifying the water elution component by using a sephadex column with a sodium chloride aqueous solution with the concentration of 0.1-0.3 mol/L as an eluent to obtain the cynomorium songaricum polysaccharide compound with the linkage mode shown in the formula V.
8. Use of a cynomorium songaricum polysaccharide compound, a pharmaceutically acceptable salt, solvate, hydrate, prodrug compound, polymorph, enantiomer or cynomorium songaricum polysaccharide of claim 5 in the preparation of a phosphodiesterase type 5 inhibitor, wherein the cynomorium songaricum polysaccharide compound has a linkage pattern represented by formula I, formula II, formula III, formula IV or formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
9. use of a cynomorium songaricum polysaccharide compound, a pharmaceutically acceptable salt, solvate, hydrate, prodrug compound, polymorph, enantiomer or cynomorium songaricum polysaccharide according to claim 5 in the manufacture of a medicament for the prevention and treatment of human or veterinary phosphodiesterase type 5 related diseases, said cynomorium songaricum polysaccharide compound having a linkage pattern of formula I, formula II, formula III, formula IV or formula V:
-3) - α -D-Araf- (1→3) - α -D-Glcp- (1→4) α -D-GalpA6Me- (1→formula I;
-alpha-Glcp- (1-6) -alpha-Glcp- (1-3) -alpha-Glcp- (1-6) -alpha-Glcp- (1-formula III;
10. the use according to claim 9, wherein the disease associated with phosphodiesterase type 5 comprises pulmonary hypertension, male erectile dysfunction, male testicular ischemia, female sexual dysfunction, dysmenorrhea, benign prostatic hyperplasia, ischemic angina, hypertension, alzheimer's disease, arteriosclerosis, stroke, arteriosclerotic occlusion, arterial embolism, chronic asthma, bronchitis, allergic asthma, allergic rhinitis, glaucoma or a disease characterized by intestinal motility disorder.
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